Contamination in genotyping of transgenic mice? - (Oct/18/2005 )

I think I encounter a stubbon contamination problem in PCR to genotype my transgenic mouse. I get 20 transgenic mouse, and use PCR to screen positive transgenic mouse. I designed a pair of primers cover the promoter and the gene I interested. That means, only positive transgenic mouse get generate PCR products. But I found 2 mouse have strong bands, and 14 others, including the wild type mouse (negative control) also have weak bands. I changed everything, and this time, no bands in negative controls, but many other mouse still have weak bands. It's impossible all these are positive transgenic mouse, so what's the problem here?

-macrosky-

QUOTE (macrosky @ Oct 18 2005, 12:45 PM)

I think I encounter a stubbon contamination problem in PCR to genotype my transgenic mouse. I get 20 transgenic mouse, and use PCR to screen positive transgenic mouse. I designed a pair of primers cover the promoter and the gene I interested. That means, only positive transgenic mouse get generate PCR products. But I found 2 mouse have strong bands, and 14 others, including the wild type mouse (negative control) also have weak bands. I changed everything, and this time, no bands in negative controls, but many other mouse still have weak bands. It's impossible all these are positive transgenic mouse, so what's the problem here?

Hi there,

Indeed, bands in the negative controls is a clear sign of possible contomination of solutions or plasticware with template DNA. The best way to get rid of contamination is to CHANGE ALL PCR REAGENTS. Always works. So, you did everything right, and if you don't see bands in your negative controls any more - - it's no longer the contamination issue. Here is one of the problems you may be dealing with: - primers bind somewhere else besides your construct; Increase the annealing temperature up to 65 (or run the gradient). The more the annealing temperature, the less inspecific priming... .It's a good idea to design two, three pairs of primers against different fragments of your construct. Test them all to check which one works better.

best!

-cska_fan-

Hi, Thanks a lot. I am trying different Tm as well as different primers.

QUOTE (cska_fan @ Oct 18 2005, 01:42 PM)

QUOTE (macrosky @ Oct 18 2005, 12:45 PM)

I think I encounter a stubbon contamination problem in PCR to genotype my transgenic mouse. I get 20 transgenic mouse, and use PCR to screen positive transgenic mouse. I designed a pair of primers cover the promoter and the gene I interested. That means, only positive transgenic mouse get generate PCR products. But I found 2 mouse have strong bands, and 14 others, including the wild type mouse (negative control) also have weak bands. I changed everything, and this time, no bands in negative controls, but many other mouse still have weak bands. It's impossible all these are positive transgenic mouse, so what's the problem here?

Hi there,

Indeed, bands in the negative controls is a clear sign of possible contomination of solutions or plasticware with template DNA. The best way to get rid of contamination is to CHANGE ALL PCR REAGENTS. Always works. So, you did everything right, and if you don't see bands in your negative controls any more - - it's no longer the contamination issue. Here is one of the problems you may be dealing with: - primers bind somewhere else besides your construct; Increase the annealing temperature up to 65 (or run the gradient). The more the annealing temperature, the less inspecific priming... .It's a good idea to design two, three pairs of primers against different fragments of your construct. Test them all to check which one works better.